Inhibitors of akt activity

ABSTRACT

The present invention is directed to compounds comprising a triazolo[4,3-b]pyridazine moiety which inhibit the activity of Akt, a serine/threonine protein kinase. The invention is further directed to chemotherapeutic compositions containing the compounds of this invention and methods for treating cancer comprising administration of the compounds of the invention.

BACKGROUND OF THE INVENTION

[0001] The present invention-relates to quinazoline containing compoundsthat are inhibitors of the activity of one or more of the isoforms ofthe serine/threonine kinase, Akt (also known as PKB). The presentinvention also relates to pharmaceutical compositions comprising suchcompounds and methods of using the instant compounds in the treatment ofcancer.

[0002] Apoptosis (programmed cell death) plays essential roles inembryonic development and pathogenesis of various diseases, such asdegenerative neuronal diseases, cardiovascular diseases and cancer.Recent work has led to the identification of various pro- andanti-apoptotic gene products that are involved in the regulation orexecution of programmed cell death. Expression of anti-apoptotic genes,such as Bcl2 or Bcl-x_(L), inhibits apoptotic cell death induced byvarious stimuli. On the other hand, expression of pro-apoptotic genes,such as Bax or Bad, leads to programmed cell death (Aams et al. Science,281:1322-1326 (1999)). The execution of, programmed cell death ismediated by caspase −1 related proteinases, including caspase-3,caspase-7, caspase-8 and caspase-9 etc (Thornberry et al. Science,281:1312-1316 (1998)).

[0003] The phosphatidylinositol 3′-OH kinase (PI3K)/Akt/PKB pathwayappears important for regulating cell survival/cell death (Kulik et al.Mol. Cell. Bibl. 17:1595-1606 (1997); Franke et al, Cell, 88:435-437(1997); Kauffmann-Zeh et al. Nature 385:544-548 (1997) Hemmings Science,275:628-630 (1997); Dudek et al., Science, 275:661-665 (1997)). Survivalfactors, such as platelet derived growth factor (PDGF), nerve growthfactor (NGF) and insulin-like growth factor-1 (IGF-1), promote cellsurvival under various conditions by inducing the activity of PI3K(Kulik et al. 1997, Hemmings 1997). Activated PI3K leads to theproduction of phosphatidylinositol (3,4,5)-triphosphate(PtdIns(3,4,5)-P3), which in turn binds to, and promotes the activationof, the serine/threonine kinase Akt, which contains a pleckstrinhomology (PH)-domain (Franke et al Cell, 81:727-736 (1995); HemmingsScience, 277:534 (1997); Downward, Curr. Opin. Cell Biol. 10:262-267(1998), Alessi et al., EMBO J. 15: 6541-6551 (1996)). Specificinhibitors of PI3K or dominant negative Akt/PKB mutants abolishsurvival-promoting activities of these growth factors or cytokines. Ithas been previously disclosed that inhibitors of PI3K (LY294002 orwortmannin) blocked the activation of Akt/PKB by upstream kinases. Inaddition, introduction of constitutively active PI3K or Akt/PKB mutantspromotes cell survival under conditions in which cells normally undergoapoptotic cell death (Kulik et al. 1997, Dudek et al. 1997).

[0004] Analysis of Akt levels in human tumors showed that Akt2 isoverexpressed in a significant number of ovarian (J. Q. Cheung et al.Proc. Natl. Acad. Sci. U.S.A. 89:9267-9271 (1992)) and pancreaticcancers (J. Q. Cheung et al. Proc. Natl. Acad. Sci. U.S.A. 93-3636-3641(1996)). Similarly, Akt3 was found to be overexpressed in breast andprostate cancer cell lines (Nakatani et al. J. Biol. Chem.274:21528-21532 (1999).

[0005] The tumor suppressor PTEN, a protein and lipid phosphatase thatspecifically removes the 3′ phosphate of PtdIns(3,4,5)-P3, is a negativeregulator of the PI3K/Akt pathway (Li et al. Science 275:1943-1947(1997), Stambolic et al. Cell 95:29-39 (1998), Sun et al. Proc. Natl.Acad. Sci. U.S.A. 96:6199-6204 (1999)). Germline mutations of PTEN areresponsible for human cancer syndromes such as Cowden disease (Liaw etal. Nature Genetics 16:64-67 (1997)). PTEN is deleted in a largepercentage of human tumors and tumor cell lines without functional PTENshow, elevated levels of activated Akt (Li et al. supra, Guldberg et al.Cancer Research 57:3660-3663 (1997), Risinger et al. Cancer Research57:4736-4738 (1997)).

[0006] These observations demonstrate that the PI3K/Akt pathway playsimportant roles for regulating cell survival or apoptosis intumorigenesis.

[0007] Three members of the Akt/PKB subfamily of second-messengerregulated serine/threonine protein kinases have been identified andtermed Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ respectively. The isoformsare homologous, particularly in regions encoding the catalytic domains.Akt/PKBs are activated by phosphorylation events occurring in responseto PI3K signaling. PI3K phosphorylates membrane inositol phospholipids,generating the second messengers phosphatidylinositol3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, whichhave been shown to bind to the PH domain of Akt/PKB. The current modelof Akt/PKB activation proposes recruitment of the enzyme to the membraneby 3′-phosphorylated phosphoinositides, where phosphorylation of theregulatory sites of Akt/PKB by the upstream kinases occurs (B. A.Hemmings, Science 275:628-630 (1997); B. A. Hemmings, Science 276:534(1997); J. Downward, Science 279:673-674 (1998)).

[0008] Phosphorylation of Akt1/PKBα occurs on two regulatory sites,Thr208 in the catalytic domain activation loop and on Ser⁴⁷³ near thecarboxy terminus (D. R. Alessi et al; EMBO J. 15:6541-6551 (1996) and R.Meier et al. J. Biol. Chem 272:30491-30497 (1997)). Equivalentregulatory phosphorylation sites occur in Akt2/PKKBβ and Akt3/PKBγ. Theupstream kinase, which phosphorylates Akt/PKB at the activation loopsite has been cloned and termed 3′-phosphoinositide dependent, proteinkinase 1 (PDK1). PDK1 phosphorylates not only Akt/PKB, but also p70ribosomal S6 kinase, p90RSK, serum and glucocorticoid-regulated kinase(SGK), and protein kinase C. The upstream kinase phosphorylating theregulatory site of Akt/PKB near the carboxy terminus has not beenidentified yet, but recent reports imply a role for the integrin-linkedkinase (ILK-1), a serine/threonine protein kinase, orautophosphorylation.

[0009] Inhibition of Akt-activation and activity can be achieved byinhibiting PI3K with inhibitors such as LY294002 and wortmannin.However, PI3K inhibition has the potential to indiscriminately affectnot just all three Akt isozymes but also other PH domain-containingsignaling molecules that are dependent on PdtIns(3,4,5)P3, such as theTec family of tyrosine kinases. Furthermore, it has been disclosed thatAkt can be activated by growth signals that are independent of PI3K.

[0010] Alternatively, Akt activity can be inhibited by blocking theactivity of the upstream kinase PDK1. No specific PDK1 inhibitors havebeen disclosed. Again, inhibition of PDK1 would result in inhibition ofmultiple protein kinases whose activities depend on PDK1, such asatypical PKC isoforms, SGK, and S6 kinases (Williams et al. Curr. Biol.10:439-448 (2000).

[0011] It is an object of the instant invention to provide novelcompounds, that are inhibitors of Akt/PKB.

[0012] It is also an object of the present invention to providepharmaceutical compositions that comprise.

[0013] It is also an object of the present invention to provide a methodfor treating cancer that comprises administering such inhibitors ofAkt/PKB activity.

SUMMARY OF THE INVENTION

[0014] The instant invention provides for compounds that inhibit ofAkt/PKB activity. In particular, the compounds disclosed selectivelyinhibit one or two of the Akt/PKB isoforms. The invention also providesfor compositions comprising such inhibitory compounds and methods ofinhibiting Akt/PKB activity by administering the compound to a patientin need of treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The compounds of the instant invention are useful in theinhibition of the activity of the serine/threonine kinase Akt. In afirst embodiment of this invention, the inhibitors of Akt activity areillustrated by the formula A:

[0016] wherein

[0017] R¹ represents phenyl, furyl, thienyl or pyridinyl, any of whichgroups may be optionally substituted with one, two or threesubstituents, independently selected from:

[0018] a) halogen;

[0019] b) C₁₋₄ alkyl;

[0020] c) C₁₋₄ alkoxy;

[0021] d) cyano;

[0022] e) di(C₁₋₄ alkyl)amino;

[0023] f) hydroxy;

[0024] R² represents amino-C₁-₆ alkyl, C₁₋₄ alkylamino-(C₁₋₆)alkyl,di(C₁₋₄ alkyl)amino-(C₁₋₆)alkyl, hydroxy-(C₁₋₆)alkyl or C₁₋₄alkoxy-(C₁₋₆)alkyl, any of which groups may be optionally substituted;

[0025] R³represents hydrogen or C₁₋₆alkyl; and

[0026] R⁴ independently represents hydrogen, C₁₋₆-alkyl, halogen, HO— orC₁₋₆ alkyl-O;

[0027] r is 0, 1 or 2;

[0028] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0029] In another embodiment the inhibitors of the instant invention areillustrated by the formula A-I:

[0030] wherein

[0031] R² is as defined with reference to formula A above;

[0032] R⁴ is selected from: C₃₋₇ cycloalkyl and phenyl, any of whichgroups may be optionally substituted;

[0033] r is 0, 1 or 2;

[0034] s is 0; 1, 2 or 3; and

[0035] R⁵ independently represents halogen, C₁₋₄ alkyl or C₁₋₆ alkoxy;

[0036] or the pharmaceutically acceptable salts thereof.

[0037] Specific compounds of the instant invention include:

[0038]2,2,N,N-tetramethyl-N-(3-phenyl-[1,2,4]triazolo[3,4-α]phthalazin-6-yl)-propane-1,3-diamine

[0039]N′-[3-(4-Methoxy-phenyl)-[1,2,4]triazolo[4,3-α]phthalazin-6-yl)-2,2,N,N-tetramethylpropane-1,3-diamine

[0040] or a pharmaceutically acceptable salt thereof.

[0041] As used herein, the expression “C₁₋₆ alkyl” includes methyl andethyl groups, and straight-chained or branched propyl, butyl, pentyl andhexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl,isopropyl, tert-butyl and 2,2-dimethylpropyl. Derived expressions suchas “C₁₋₆ alkoxy” are to be construed accordingly.

[0042] As used herein, the expression “C₁₋₄ alkyl” includes methyl andethyl groups, and straight-chained or branched propyl and butyl groups.Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl andtert-butyl. Derived expressions such as “C₁₋₄ alkoxy” are to beconstrued accordingly.

[0043] Typical C₃₋₇ cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

[0044] The expression “C₃₋₇ cycloalkyl(C₁₋₆)alkyl” as used hereinincludes cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl andcyclohexylmethyl.

[0045] Typical C₄₋₇ cycloalkenyl groups include cyclobutenyl,cyclopentenyl and cyclohexenyl.

[0046] Typical aryl groups include phenyl and naphthyl, preferablyphenyl.

[0047] The expression “aryl(C₁₋₆)alkyl” as used herein includes benzyl,phenylethyl, phenylpropyl and naphthylmethyl.

[0048] The term “halogen” as used herein includes fluorine, chlorine,bromine and iodine, especially fluorine or chlorine.

[0049] In a particular embodiment, R² represents amino-C₁₋₆ alkyl, C₁₋₄alkylamino-(C₁₋₆)alkyl or di(C₁₋₄ alkyl)amino-(C₁₋₆ alkyl.Representative values of R² include but are not limited todimethylaminomethyl, aminoethyl, dimethylaminoethyl, diethylaminoethyl,3-dimethylaminopropyl, 3-methylaminopropyl,3-dimethylamino-2,2-dimethylpropyl and, 3-dimethylamino-2-methylpropyl.

[0050] For use in medicine, the salts of the compounds of formula I willbe pharmaceutically acceptable salts. Other salts may, however, beuseful in the preparation of the compounds according to the invention orof their pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts of the compounds of this invention include acidaddition salts which may, for example, be formed by mixing a solution ofthe compound according to the invention with a solution of apharmaceutically acceptable acid such as hydrochloric acid, sulphuricacid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid,acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid,carbonic acid or phosphoric acid. Furthermore, where the compounds ofthe invention carry an acidic moiety, suitable pharmaceuticallyacceptable salts thereof may include alkali metal salts, e.g. sodium orpotassium salts; alkaline earth metal salts, e.g. calcium or magnesiumsalts; and salts formed with suitable organic ligands, e.g. quaternaryammonium salts.

[0051] The present invention includes within its scope prodrugs of thecompounds of formula I above. In general, such prodrugs will befunctional derivatives of the compounds of formula I which are readilyconvertible in vivo into the required compound of formula I.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in Design of Prodrugs,ed. H. Bundgaard, Elsevier, 1985.

[0052] Where the compounds according to the invention have at least oneasymmetric center, they may accordingly exist as enantiomers. Where thecompounds according to the invention possess two or more asymmetriccenters, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present invention.

[0053] The compounds of the instant invention are inhibitors of theactivity of Akt and are thus useful in the treatment of cancer, inparticular cancers associated with irregularities in the activity of Aktand/or GSK3. Such cancers include, but are not limited to ovarian,pancreatic and breast cancer.

[0054] In an embodiment of the invention, the instant compound is aselective inhibitor whose inhibitory efficacy is dependent on the PHdomain. In this embodiment, the compound exhibits a decrease in in vitroinhibitory activity or no in vitro inhibitory activity against truncatedAkt proteins lacking the PH domain.

[0055] In another embodiment of the invention, the instant compound is aselective inhibitor whose inhibitory efficacy is dependent on the regionof the proteins between the PH domain and the kinase domain. (SeeKonishi et al. Biochem. and Biophys. Res. Comm. 216: 526-534 (1995),FIG. 2) That region will be referred to as the hinge region. In thisembodiment, the compound exhibits a decrease in in vitro inhibitoryactivity or no in vitro inhibitory activity against truncated Aktproteins lacking the PH domain and the hinge region.

[0056] Such an inhibitor that is dependent on either the PH domain, thehinge region or both provides a particular advantage since the PHdomains and hinge regions in the three Akt isoforms lack the sequencehomology that is present in the rest of the protein, particularly thehomology found in the kinase domains (which comprise the catalyticdomains and ATP-binding consensus sequences). It is there fore observedthat certain inhibitor compounds, such as those described herein, arenot only selective for one or two isoforms of Akt, but also are weakinhibitors or fail to inhibit other kinases, such as PKA and PKC, whosekinase domains share some sequence homology with the kinase domains ofthe Akt/PKB isoforms. Both PKA and PKC lack a PH domain.

[0057] In a further embodiment, the instant compound is selected fromthe group of a selective inhibitor of Akt 1, a selective inhibitor ofAkt 2 and a selective inhibitor of both Akt 1 and Akt 2.

[0058] In another embodiment, the instant compound is selected from thegroup of a selective inhibitor of Akt 1, a selective inhibitor of Akt 2,a selective inhibitor of Akt3 and a selective inhibitor of two of thethree Akt isoforms.

[0059] In another embodiment, the instant compound is a selectiveinhibitor of call-three Akt isoforms, but is not an inhibitor of one,two or all of such Akt isoforms that have been modified to delete the PHdomain, the hinge region or both the PH domain and the hinge region.

[0060] The present invention is further directed to a method ofinhibiting Akt activity which comprises administering to a mammal inneed thereof a pharmaceutically effective amount of the instantcompound.

[0061] The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers, excipients or diluents, in apharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally,including the intravenous, intramuscular, intraperitoneal, subcutaneous,rectal and topical routes of administration.

[0062] The pharmaceutical compositions containing the active ingredientmay be in a form suitable for oral use, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups or elixirs.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example,microcrystalline cellulose, sodium crosscarmellose, corn starch, oralginic acid; binding agents, for example starch, gelatin,polyvinyl-pyrrolidone or acacia, and lubricating agents, for example,magnesium stearate, stearic acid or talc: The tablets may be uncoated orthey may be coated by known techniques to mask the unpleasant taste ofthe drug or delay disintegration and absorption in the gastrointestinaltract and thereby provide a sustained action over a longer period. Forexample, a water soluble taste masking material-such ashydroxypropylmethyl-cellulose or hydroxypropyl-cellulose, or a timedelay material such as ethyl cellulose, cellulose acetate buryrate maybe employed.

[0063] Formulations for oral use may also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater soluble carrier such as polyethyl-eneglycol or an oil medium, forexample peanut oil, liquid paraffin, or olive oil.

[0064] Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitol monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

[0065] Oily suspensions may be formulated by suspending the activeingredient in a vegetable oil, for example arachis oil, olive oil,sesame oil or coconut oil, or in mineral oil such as liquid paraffin.The oily suspensions may contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents such as thoseset forth above, and flavoring agents may be added to provide apalatable oral preparation. These compositions may be preserved by theaddition of an anti-oxidant such as butylated hydroxyanisol oralpha-tocopherol.

[0066] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present. These compositions may bepreserved by the addition of an anti-oxidant such as ascorbic acid.

[0067] The pharmaceutical compositions of the invention may also be inthe form of an oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, for exampleliquid paraffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring agents, preservatives and antioxidants.

[0068] Syrups and elixirs may be formulated with sweetening agents, forexample glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also, contain a demulcent, a preservative, flavoringand coloring agents and antioxidant.

[0069] The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

[0070] The sterile injectable preparation may also be a sterileinjectable oil-in-water microemulsion where the active ingredient isdissolved in the oily phase. For example, the active ingredient may befirst dissolved in a mixture of soybean oil and lecithin. The oilsolution then introduced into a water and glycerol mixture and processedto form a microemulation.

[0071] The injectable solutions or microemulsions may be introduced intoa patient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

[0072] The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

[0073] Compounds of Formula A may also be administered in the form of asuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal-temperature and will therefore melt in the rectum to release thedrug. Such materials include cocoa butter, glycerinated, gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

[0074] For topical use, creams, ointments, jellies, solutions orsuspensions, etc:, containing the compound of Formula A are employed.(For purposes of this application, topical application shall includemouth washes and gargles.)

[0075] The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen.

[0076] As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

[0077] The instant compounds may also be co-administered with other wellknown therapeutic agents that are selected for their particularusefulness against the condition that is being treated. For example, theinstant compounds may be useful in combination with known anti-cancerand cytotoxic agents. Similarly, the instant compounds may be useful incombination with agents that are effective in the treatment andprevention of neurofibromatosis, restinosis, polycystic kidney disease,infections of hepatitis delta and related viruses and fungal infections.The instant compositions may also be useful in combination with otherinhibitors of parts of the signaling pathway that links cell surfacegrowth factor receptors to nuclear signals initiating cellularproliferation. Thus, the instant compounds may be utilized incombination with inhibitors of prenyl-protein transferase, includingprotein substrate competitive inhibitors of farnesyl-proteintransferase, farnesyl pyrophosphate competitive inhibitors of theactivity of farnesyl-protein transferase and/or inhibitors ofgeranylgeranyl-protein transferase. The instant compositions may also beco-administered with compounds that are selective inhibitors ofgeranylgeranyl protein transferase or selective inhibitors offarnesyl-protein transferase. The instant compositions may also beadministered in combination with a compound that has Raf antagonistactivity.

[0078] The compounds of the instant invention may also beco-administered with other well known cancer therapeutic agents that areselected for their particular usefulness against the condition that isbeing treated. Included in such combinations of therapeutic agents arecombinations with an antineo-plastic agent. It is also understood thatthe instant compositions and combinations may be used in conjunctionwith other methods of treating cancer and/or tumors, including radiationtherapy and surgery.

[0079] Additionally, compositions of the instant invention may also beuseful as radiation sensitizers. For instance, radiation therapy,including x-rays or gamma rays that are delivered from either anexternally applied beam or by implantation of tiny radioactive sources,may used in combination with the instant compounds to treat cancer.

[0080] If formulated as a fixed dose, such combination products employthe combinations of this invention within the dosage range describedbelow and the other pharmaceutically active agent(s) within its approveddosage range. Combinations of the instant invention may alternatively beused sequentially with known pharmaceutically acceptable agent(s) when amultiple combination formulation is inappropriate.

[0081] The instant compositions may also be useful in combination withan integrin antagonist for the treatment of cancer, as described in U.S.Ser. No. 09/055,487, filed Apr. 6, 1998, which is incorporated herein byreference.

[0082] As used herein the term an integrin antagonist refers tocompounds which selectively antagonize, inhibit or counteract binding ofa physiological ligand to an integrin(s) that is involved in theregulation of angiogenisis, or in the growth and invasiveness of tumorcells. In particular, the term refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe αvβ3 integrin, which selectively antagonize, inhibit or counteractbinding of a physiological-ligand to the αvβ5 integrin, whichantagonize, inhibit or counteract binding of a physiological ligand toboth the αvβ3 integrin and the αvβ5 integrin, or which antagonize,inhibit or counteract the activity of the particular integrin(s)expressed on capillary endothelial cells. The term also refers toantagonists of the αvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1 and α6β4integrins. The term also refers to antagonists of any combination ofαvβ3, αvβ5, αvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1 and α6β4 integrins. Theinstant compounds may also be useful with other agents that inhibitangiogenisis and thereby inhibit the growth and invasiveness of tumorcells, including, but not limited to angiostatin and endostatin.

[0083] When a composition according to this invention is administeredinto a human subject, the daily dosage will normally be determined bythe prescribing physician with the dosage generally-varying according tothe age, weight, and response of the individual patient, as well as theseverity of the patient's symptoms.

[0084] In one exemplary application, a suitable amount of an inhibitorof one or two of the Akt/PKB isoforms is administered to a mammalundergoing treatment for cancer. Administration occurs in an amount ofinhibitor of between about 0.1 mg/kg of body weight to about 60 mg/kg ofbody weight per day, preferably of between 0.5 mg/kg of body weight toabout 40 mg/kg of body weight per day. A particular therapeutic dosagethat comprises the instant composition includes from about 0.01 mg toabout 1000 mg of inhibitor of one or two of the Akt/PKB isoforms.Preferably, the dosage comprises from about 1 mg to about 1000 mg ofinhibitor of one or two of the Akt/PKB isoforms.

[0085] Examples of an antineoplastic agent include, in general,microtubule-stabilising agents (such as paclitaxel (also known asTaxol®), docetaxel (also known as Taxotere®), or their derivatives);alkylating agents, anti-metabolites; epidophyllotoxin; an antineoplasticenzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinumcoordination complexes; biological response modifiers and growthinhibitors; hormonal/anti-hormonal therapeutic agents and haematopoieticgrowth factors.

[0086] Example classes of antineoplastic agents include, for example,the anthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the taxanes, the epothilones,discodermolide, the pteridine family of drugs, diynenes and thepodophyllotoxins. Particularly useful members of those classes include,for example, doxorubicin, carminomycin, daunorubicin, aminopterin,methotrexate, methopterin, dichloro-methotrexate, mitomycin C,porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosinearabinoside, podophyllotoxin or podo-phyllotoxin derivatives such asetoposide, etoposide phosphate or teniposide, melphalan, vinblastine,vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like.Other useful antineoplastic agents include estramustine, cisplatin,carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosamide,melphalan; hexamethyl melamine, thiotepa, cytarabin, idatrexate,trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11,topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindolederivatives, interferons and interleukins.

[0087] All patents, publications and pending patent applicationsidentified are hereby incorporated by reference.

[0088] Abbreviations used in the description of the chemistry and in theExamples that follow are:

[0089] Ac₂O Acetic anhydride;

[0090] Boc t-Butoxycarbonyl;

[0091] DBU 1,8-diazabicyclo[5,4,0]undec-7-ene;

[0092] Reactions used to generate the compounds of this invention areprepared by employing reactions as shown in the Scheme 1, in addition toother standard manipulations such as ester hydrolysis, cleavage ofprotecting groups, etc., as may be known in the literature orexemplified in the experimental procedures. Substituents R, R^(a) andR^(b), as shown in the Scheme, represent the substituents R¹ and R²;however their point of attachment to the ring is illustrative only andis not meant to be limiting.

[0093] These reactions may be employed in a linear sequence to providethe compounds of the invention or they may be used to synthesizefragments which are subsequently joined by the alkylation reactionsdescribed in the Schemes.

[0094] SYNOPSIS OF SCHEME 1:

[0095] The requisite intermediates are in some cases commerciallyavailable, or can be prepared according to literature procedures. Thusas shown in Scheme 1, a suitably substituted 1,4-dichlorophthalazine isreacted with a stoichemetric amount of hydrazine to provide the1-chloro-4-hydrazinophthalazine I, which can-then undergoacylation/cyclization with an appropriate acid chloride to provide thethiazolophthalazine II. The intermediate II can then react with asuitable amine to provide the instant compound III

EXAMPLES

[0096] Examples provided are intended to assist in a furtherunderstanding of the invention. Particular materials employed, speciesand conditions are intended to be further illustrative of the inventionand not limitative of the reasonable scope thereof

Example 12,2,N,N-tetramethyl-N-(3-phenyl-[1,2,4]triazolo[3,4-α]phthalazin-6-yl)propane-1,3-diamine(Compound 1)

[0097] Step 1: 1-Chloro-4-hydrazinophthalazine Hydrochloride

[0098] To a stirred solution of hydrazine hydrate (40 ml) in ethanol(120 mL) at 80° C. was added 1,4-dichlorophthalazine (20 g). Thisreaction, mixture was stirred at 80° C. for 0.5 hours, then left to cooland the product was collected by filtration and dried under vacuum togive 1-chloro-4-hydrazinophthalazine hydrochloride (14.6 g). ¹H NMR (250MHz, DMSO)•4.64 (2H, vbs), 7.2 (1H, vbs), 7.92 (4H, bm).

[0099] Step 2: 6-Chloro-3-phenyl-1,2,4-triazolo[3,4-a]phthalazine

[0100] To a solution of 1-chloro-4-hydrazinophthalazine hydrochloride(10 g) in dioxan (220 ml) was added triethylamine (7.24 ml) and benzoylchloride (6.04 ml). This mixture was heated at reflux for 8 hours undernitrogen. After cooling the reaction mixture was concentrated undervacuum and the solid obtained was collected by filtration, washed withwater and diethyl ether and dried under vacuum, to yield the titlecompound (12.0 g). ¹H NMR (250 MHz, DMSO)•7.60 (3H, m), 8.00 (1H, t,J=8.4 Hz), 8.19 (1H, t, J=8.4 Hz), 8.31 (3H, m), 8.61 (1H, d, J=6.3 Hz).

[0101] Step 3:2,2,N,N-tetramethyl-N-(3-phenyl-[1,2,4]triazolo[3,4-α]phthalazin-6yl)-propane-1,3-diamine

[0102] The title compound was prepared as described in Example 1, Step3, but replacing the6-Chloro-7-cyclobutyl-3-phenyl-[1,2,4]triazolo[4,3-b]pyridazine with the6-Chloro-3phenyl-1,2,4-triazolo[3,4-a]phthalazine from Step 2.

[0103]¹H (360 MHz, CDCl₃) δ 1.13 (6H, s), 2.35 (2H, s), 2.46-2.50 (8H,m), 3.47 (2H, vbs), 7.16-7.27 (2H, m), 7.44-7.86 (5H, m), 8.55-8.57 (2H,m), 8.68 (1H, m). MS (ES+) MH⁺=375

Example 2N′-[3-(4-Methoxy-phenyl)-[1,2,4]triazolo[4,3-α]phthalazin-6-yl)-2,2,N,N-tetramethyl-propane-1,3-diamine(Compound 2)

[0104] The title compound was prepared in an analogous fashion toExample 1, except substituting 3-fluorobenzoic hydrazine for the benzoichydrazine in Step 2. ¹H NMR (360 MHz, CDCl₃) δ 1.13 (6H, s), 2.45 (6H,s), 2.49 (2H, s), 3.45-3.46 (2H, m), 3.90 (3H, s) 7.04-7.07 (2H, m),7.65-7.70 (2H, m), 7.80-7.84 (1H, m), 8.51 (2H, m), 8.66 (1H, m). MS(ES+) MH⁺=405

Example 3

[0105] Cloning of the Human Akt Isoforms and ΔPH-Akt1

[0106] The pS2neo vector (deposited in the ATCC on Apr. 3, 2001 as ATCC)was prepared a's follows: The pRmHA3 vector (prepared as described inNucl. Acid Res. 16:1043-1061 (1988)) was cut with BglII and a 2734 bpfragment was isolated. The pUChsneo vector (prepared as described inEMBO J. 4:167-171 (1985)) was also cut with BglII and a 4029 bp band wasisolated. These two isolated fragments were ligated together to generatea vector termed pS2neo-1. This plasmid contains a polylinker between ametallothionine promoter and an alcohol dehydrogenase poly A additionsite. It also has a neo resistance gene driven by a heat shock promoter.The pS2neo-1 vector was cut with Psp5II and BsiWI. Two complementaryoligonucleotides were synthesized and then annealed (CTGCGGCCGC(SEQ.ID.NO.: 1) and GTACGCGGCCGCAG (SEQ.ID.NO.: 2)). The cut pS2neo-1and the annealed oligonucleotides were ligated together to generate asecond vector, pS2neo. Added in this conversion was a NotI site to aidin the linearization prior to transfection into S2 cells.

[0107] Human Akt1 gene was amplified by PCR (Clontech) out of a humanspleen cDNA (Clontech) using the 5′primer:5′CGCGAATTCAGATCTACCASTEAGCGACGTGGCTATTGTG-3′ (SEQ.ID.NO.: 3), and the3′ primer: 5′CGCTCTAGAGGATCCTCAGGCCGTGCTGCTGGC3′ (SEQ.ID.NO.: 4). The5′primer included an EcoRI and BglII site. The 3′ primer included anXbaI and BamHI site for cloning purposes. The resultant PCR product wassubcloned into pGEM3Z (Promega) as an EcoRI/Xba I fragment. Forexpression/purification purposes, a middle T tag was added to the 5′ endof the full length Akt1 gene using the PCR primer:5′GTACGATGCTGAACGATATCTTCG 3′ (SEQ.ID.NO.: 5). The resulting PCR productencompassed a 5′ KpnI site and a 3′ BamHI site which were used tosubclone the fragment in frame with a biotin tag containing insect cellexpression vector, pS2neo.

[0108] For the expression of a pleckstrin homology domain (PH) deleted(Δ aa 4-129, which includes deletion of a portion of the Akt1 hingeregion) version of Akt1. PCR deletion mutagenesis was done, using thefull length Akt1 gene in the pS2neo vector as template. The PCR wascarried out in 2 steps using overlapping internal primers(5′GAATACATGCCGATGGAAAGCGACΔGGGGCTGAAGAGATGGA GGTG 3′ (SEQ.ID:NO.: 6),and 5′ CCCCTCCATCTCTTCAGCCCCΔGTCGC TTTCCATCGGCATGTATTC 3′ (SEQ.ID.NO.:7)) which encompassed the deletion and 5′ and 3′ flanking primers whichencompassed the KpnI site and middle T tag on the 5′ end. The final PCRproduct was digested with KpnI and SmaI and ligated into the pS2neo fulllength, Akt1 KpnI/Sma I cut vector, effectively replacing the 5′ end ofthe clone with the deleted version.

[0109] Human Akt3 gene was amplified by PCR of adult brain cDNA(Clontech) using the amino terminal oligo primer: 5′GAATTCAGATCTACCATGAGCGATGTTACCATTGTG 3′ (SEQ.ID.NO.: 8); and the carboxyterminal oligo primer: 5′ TCTAGATCTTATTCTCGTCCACTTGCAGAG 3′(SEQ.ID.NO.:9).

[0110] These primers included a 5′ EcoRI/BglII site and a 3′ XbaI/BglIIsite for cloning purposes. The resultant PCR product was cloned into theEcoRI and XbaI sites of pGEM4Z (Promega). For expression/purificationpurposes, a middle T tag was added to the 5′ end of the full length Akt3clone using the PCR primer: 5′GGTACCATGGAATACATGCCGATGGAAAGCGATGTTACCATTGTGAAG 3′(SEQ.ID.NO.: 10). Theresultant PCR product encompassed a 5′ KpnI site which allowed in framecloning with the biotin tag containing insect cell expression vector,pS2neo.

[0111] Human Akt2 gene was amplified by PCR from human thymus cDNA(Clontech) using the amino terminal oligo primer: 5′AAGCTTAGATCTACCATGAATGAGGTGTCTGTC 3′ (SEQ.ID.NO.: 11); and the carboxyterminal oligo primer: 5′ GAATTCGGATCCTCACTCGCGGATGCTGGC 3′ (SEQ.ID.NO.:12). These primers included a 5′ HindIII/BglII site and a 3′ EcoR/IBamHI site for cloning purposes. The resultant PCR product was subclonedinto the HindIII/EcoRI sites of pGem3Z (Promega). Forexpression/purification purposes, a middle T tag was added to the 5′ endof the full length Akt2 using the PCR primer: 5′GGTACCATGGAATACATGCCGATGGAAAATGAGGTGTCTGTCATCAAAG 3′ (SEQ.ID.NO.: 13).The resultant PCR product was subcloned into the pS2neo vector asdescribed above.

Example 4

[0112] Expression of Human Akt Isoforms and ΔPH-Akt1

[0113] The DNA containing the cloned Akt1, Akt2, Akt3 and ΔPH-Akt1 genesin the pS2neo expression vector was purified and used to transfectDrosophila S2 cells (ATCC) by the calcium phosphate method. Pools ofantibiotic (G418, 500 μg/ml) resistant cells were selected. Cell wereexpanded to a 1.0 L volume (˜7.0×10⁶/ml), biotin and CuSO₄ were added toa final concentration of 50 μM and 50 mM respectively. Cells were grownfor 72h at 27° C. and harvested by centrifugation. The cell paste wasfrozen at −70° C. until needed.

Example 5

[0114] Purification of Human Akt Isoforms and ΔPH-Akt1

[0115] Cell paste from one liter of S2 cells, described in Example 13,was lysed by sonication with 50 mls 1% CHAPS in buffer A: (50 mM Tris pH7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM AEBSF, 10 μg/ml benzamidine, 5 μg/mlof leupeptin, aprotinin and pepstatin each, 10% glycerol and 1 mM DTT).The soluble fraction was purified on a Protein G Sepharose fast flow(Pharmacia) column loaded with 9 mg/ml anti-middle T monoclonal antibodyand eluted with 75 μM EYMPME (SEQ.ID.NO.: 14) peptide in buffer Acontaining 25% glycerol: Akt/PKB containing fractions were pooled andthe protein purity evaluated by SDS-PAGE. The purified protein wasquantitated using a standard Bradford protocol. Purified protein wasflash frozen on liquid nitrogen and stored at −70° C.

Example 6.

[0116] Kinase Assays

[0117] This procedure describes a kinase assay which measuresphosphorylation of a biotinylated GSK3-derived peptide by humanrecombinant active Akt/PBK isoforms or Akt/PBK mutants. The ³³P-labeledbiotinylated product can be captured and detected using Streptavidincoated Flashplates (NEN LifeSciences) or Streptavidin Membrane FilterPlates (Promega). Alternatively, a GSK3-derived peptide with 2 addedlysine residues was used as the substrate and subsequently capturedusing Phosphocellulose Membrane Filter Plates (Polyfiltronics).

[0118] Materials:

[0119] Active human Akt: The following active human Akt isoforms wereutilized in the in vitro assays: active human Akt1 (obtained fromUpstate Biotechnology, catalog no. 14-276; 15 μg/37 μl (6.76 μM)) orrecombinant lipid activated-Akt1 (prepared as described in Example 5);Akt2 (prepared as described in Example 5); Akt3 (prepared, as describedin Example 5); and, delta PH-Akt1 (prepared as described in Example 5).

[0120] Akt specific peptide substrate: GSK3α (S21) Peptide #3928,btion-GGRARTSSFAEPG (SEQ.ID.NO.: 15), FW=1517.8 (obtained fromMacromolecular Resources) for Streptavidin Flashplate or StreptavidinFilter Plate detection.

[0121] GSK3α (S21) Peptide #G80613, KKGGRARTSSFAEPG (SEQ.ID:NO.: 14),FW=1547.8 (obtained from Research Genetics) for Phosphocellulose filterplate detection.

[0122] Standard Assay Solutions:

[0123] A. 10× Assay Buffer: 500 mM HEPES, pH 7.5

[0124] 1% PEG

[0125] 1 mM EDTA

[0126] 1 mM EGTA

[0127] 20 mM β-Glycerol phosphate

[0128] B. Active Akt (500 nM): Diluent (1× Assay buffer, 10% glycerol,0.1% β-mercaptoethanol, 1.0 μM microcystin LR and 1.0 mM EDTA) was addedto a vial containing 37 μl of active Akt isoform (6.76 μM). Aliquotswere flash frozen in liquid N₂ and stored at −70•C.

[0129] C. 1 mM Akt specific peptide substrate in 50 mM Tris pH 7.5, 1 mMDTT.

[0130] D. 100 mM DTT in di H₂O.

[0131] E. 100× Protease Inhibitor Cocktail (PIC): 1 mg/ml benzamidine,0.5 mg/ml pepstatin, 0.5 mg/ml leupeptin, 0.5 mg/ml aprotinin.

[0132] F. 3 mM ATP, 200 mM MgCl₂ in H₂O, pH 7.9.

[0133] G. 50% (v/v) Glycerol.

[0134] H. 1% (wt/v) BSA (10 mg/ml) in diH₂O, 0.02% (w/v) NaN₃.

[0135] I. 125 mM EDTA.

[0136] J. 0.75% (wt/v) Phosphoric Acid.

[0137] K. 2.5 M Potassium Chloride.

[0138] L: Tris Buffered Saline. (TBS), 25 mM Tris, 0.15 M SodiumChloride, pH 7.2 (BupH Tris Buffered Saline Pack, Pierce catalog no.28376).

[0139] Procedure for Streptavidin Plash Plate Assay:

[0140] Step 1:

[0141] A 1 μl solution of the test compound in 100% DMSO was added to 20μl of 2×substrate solution (20 uM GSK3 Peptide, 300 μM ATP, 20 mM MgCl₂,20 μCi/ml [γ³³P] ATP, 1×Assay Buffer, 5% glycerol, 1 mM DTT, 1× PIC,0.1% BSA and 100 mM KCl). Phosphorylation reactions were initiated byadding 19 μl of 2× Enzyme solution (6.4 nM active Akt/PKB, 1× AssayBuffer, 5% glycerol, 1 mM DTT, 1×PIC and 0.1% BSA). The reactions werethen incubated at room temperature for 45 minutes.

[0142] Step 2:

[0143] The reaction was stopped by adding 170 μl of 125 mM EDTA. 200 μlof stopped reaction was transferred to a Streptavidin Flashplate® PLUS(NEN Life Sciences, catalog no. SMP103). The plate was incubated for ≧10minutes at room temperature on a plate shaker. The contents of each wellwas aspirated, and the wells rinsed 2 times with 200 μl TBS per well.The wells were then washed 3 times for 5 minutes with 200 μl TBS perwell with the plates incubated at room temperature on a platform shakerduring wash steps.

[0144] The plates were covered with sealing tape and counted using thePackard TopCount with the appropriate settings for counting [³³P] inFlashplates.

[0145] Procedure for Streptavidin Filter Plate Assay:

[0146] Step 1:

[0147] The enzymatic reactions as described in Step 1 of theStreptavidin Flash Plate Assay above were performed.

[0148] Step 2:

[0149] The reaction was stopped by adding 20 μl of 7.5M GuanidineHydrochloride. 50 μl of the stopped reaction was transferred to theStreptavidin filter plate. (SAM²™ Biotin Capture Plate, Promega, catalogno. V7542) and the reaction was incubated on the filter for 1-2 minutesbefore applying vacuum.

[0150] The plate was then washed using a vacuum manifold as follows: 1)4×200 μl/well of 2M NaCl; 2)₆×200 μl/well of 2M NaCl with 1% H₃PO₄; 3)2×200 μl/well of diH₂ O; and 4) 2×100 μl/well of 95% Ethanol. Themembranes were then allowed to air dry completely before addingscintillant.

[0151] The bottom of the plate was sealed with white backing tape, 30μl/well of Microscint 20 (Packard Instruments, catalog no. 6013621) wasadded. The top of the plate was sealed with clear sealing tape, and theplate then counted using the Packard TopCount with the appropriatesettings for [³³P] with liquid scintillant.

[0152] Procedure for Phosphocelluose Filter Plate Assay:

[0153] Step 1:

[0154] The enzymatic reactions were performed as described in Step 1 ofthe Streptavidin. Flash Plate Assay (above) utilizing KKGGRARTSSFAEPG(SEQ.ID.No.: 16) as the substrate in place of biotin-GGRARTSSFAEPG.

[0155] Step 2:

[0156] The reaction was stopped by adding 20 μl of 0.75% H₃PO₄ 50 μl ofstopped reaction was transferred to the filter plate (UNIFILTER™,Whatman P81 Strong Cation Exchanger, White Polystyrene 96 Well Plates,Polyfiltronics, catalog no. 7700-3312) and the reaction incubated on thefilter for 1-2 minutes before applying vacuum.

[0157] The plate was then washed using a vacuum manifold as follows: 1)9×200 μl/well of 0.75% H₃PO₄; and 2) 2×200 μl/well of diH₂O. The bottomof the plate was sealed with white backing tape, then 30 μl/well ofMicroscint 20 was added. The top of the plate was sealed with clearsealing tape, and the plate counted using the Packard TopCount with theappropriate settings for [³³P] and liquid scintillant.

[0158] PKA Assay

[0159] Each individual PKA assay consists of the following components:

[0160] 1) 10 μl 5×PKA assay buffer (200 mM Tris pH7.5, 100 mM MgCl₂, 5mM 2-mercaptoethanol, 0.5 mM EDTA)

[0161] 2) 10 μl of a 50 μM stock of Kemptide (Sigma) diluted into water

[0162] 3) 10 μl ³³P-ATP (prepared by diluting 1.0 μl ³³P-ATP [10 mCi/ml]into 200 μl of a 50 μM stock of unlabeled ATP)

[0163] 4) 10 μl appropriate solvent control dilution, or inhibitordilution

[0164] 5) 10 μl of a 70 nM stock of PKA catalytic subunit (UBI catalog #14-114) diluted in 0.5 mg/ml BSA.

[0165] The final assay concentrations were 40 mM Tris pH 7.5, 20 mMMgCl₂, 1 mM 2-mercaptoethanol, 0.1 mM EDTA, 10 μM Kemptide, 10 μM³³P-ATP, 14 nM PKA and 0.1 mg/ml BSA.

[0166] Assays were assembled in 96 deep-well assay plates. Components #3and #4 were premixed and in a separate tube, a mixture containing equalvolumes of components #1, #2, and #5 was prepared. The assay reactionwas initiated by adding 30 μl of the components #1, #2, and #5 mixtureto wells containing ³³P-ATP and inhibitor. The liquid in the assay wellswas mixed and the assay reactions incubated for 20 minutes at roomtemperature. The reactions were stopped by adding 50 μl 100 mM EDTA and100 mM sodium pyrophosphate and mixing.

[0167] The enzyme reaction product (phosphorylated Kemptide) wasquantitated using p81 phosphocellulose 96 well filter plates(Millipore). Each well of a p81 filter plate was filled with 75 mMphosphoric acid. The wells were aspirated and 170 μl of 75 mM phosphoricacid was added to each well. A 30-40 μl aliquot from each stopped PKA,reaction was added to corresponding wells on the filter plate containingthe phosphoric acid. The peptide was trapped on the filter following theapplication of a vacuum. The filters were washed 5× by filling wellswith 75 mM phosphoric acid followed by aspiration. After the final wash,the filters were allowed to air dry. 30 μl scintillation fluid was addedto each well and the filters counted on a TopCount (Packard).

[0168] PKC Assay

[0169] Each PKC assay consists of the following components:

[0170] 1) 5 μl 10× PKC co-activation-buffer (2.5 mM EGTA, 4 mM CaCl₂)

[0171] 2) 10 μl 5× PKC activation buffer (1.6 mg/ml phosphatidylserine,0.16 mg/ml diacylglycerol, 100 mM Tris pH 7.5, 50 mM MgCl, 5 mM2-mercaptoethanol)

[0172] 3) 5 μl ³³P-ATP (prepared by diluting 1.0 μl ³³P-ATP [10 mCi/ml]into 100 μl of a 100 μM stock of unlabeled ATP)

[0173] 4) 10 μl of a 350 μg/ml stock of myelin basic protein (MBP, UBI)diluted in water

[0174] 5) 10 μl appropriate solvent control or inhibitor dilution

[0175] 6) 10 μl of a 50 ng/ml stock of PKC (mix of isoforms from UBIcatalog # 14-115) diluted into 0.5 mg/ml BSA

[0176] Final assay concentrations were as follows: 0.25 mM EGTA, 0.4 mMCaCl, 20 mM Tris pH 7.5, 10 mM MgCl, 1 mM 2-mercaptoethanol, 0.32 mg/mlphosphatidylserine, 0.032 mg/ml diacylglycerol, 10 μM ³³P-ATP, 70 μg/mlMBP, 10 ng/ml PKC, 0.1 mg/ml BSA.

[0177] Assays are performed using 96 deep well assay plates. In eachassay well 10 μl of solvent control or appropriate inhibitor dilutionwith 5 μl ³³P-ATP (components #5 and #3) were premixed. In a separatetube, a mixture containing equal volumes of components #1, #2, #4, and#6 was prepared. The assay reaction was initiated, by adding 35 μl ofthe components #1, #2, #4, and #6 mixture to wells containing ³³P-ATPand inhibitor. The liquid in the assay wells was thoroughly mixed andthe assay reactions incubated for 20 minutes at room temperature. Thereactions were stopped by adding 100 mM EDTA (50 μl) and 100 mM sodiumpyrophosphate (50 μl) and mixing. Phosphorylated MBP was collected onPVDF membranes in 96 well filter plates and quantitated by scintillationcounting.

[0178] The results from testing the compounds described in Examples 1-2in the assays described above are shown in Table 1: TABLE 1 GSK3 PeptideSubstrate IC₅₀(μM) Counter Akt-1 screens delta IC₅₀(μM) Akt-1 PH Akt2Akt3 PKA PKC Compound 2 3.88 >50 >50 >50 >40 >40 Compound 110.5 >50 >50 >50 >40 >40

Example 7

[0179] Cell based Assays to Determine Inhibition of Akt/PKB

[0180] Cells (for example LnCaP or a PTEN^((−/−)) (tumor cell line withactivated Akt/PKB) were plated in 100 mM dishes. When the cells wereapproximately 70 to 80% confluent, the cells were refed with 5 mls offresh media and the test compound added in solution. Controls includeduntreated cells, vehicle treated cells and cells treated with eitherLY294002 (Sigma) or wortmanin (Sigma) at 20 μM or 200 nM, respectively.The cells were incubated for 2 hrs, and the media removed, The cellswere washed with PBS, scraped and transferred to a centrifuge tube. Theywere pelleted and washed again with PBS. Finally, the cell pellet wasresuspended in lysis buffer (20 mM Tris pH8, 140 mM NaCl, 2 mM EDTA, 1%Triton, 1 mM Na Pyrophosphate, 10 mM β-Glycerol Phosphate, 10 mM NaF,0.5 mm NaVO₄, 1 μM Microsystine, and 1× Protease Inhibitor Cocktail),placed on ice for 15 minutes and gently vortexed to lyse the cells. Thelysate was spun in a Beckman tabletop ultra centrifuge at 100,000×g at4° C. for 20 min. The supernatant protein was quantitated by a standardBradford protocol (BioRad) and stored at −70° C. until needed.

[0181] Proteins were immunoprecipitated (IP) from cleared lysates asfollows: For Akt1/PKBa, lysates are mixed with Santa Cruz sc-7126 (D-17)in NETN (100 mM NaCl, 20 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP-40) andProtein A/G Agarose (Santa Cruz sc-2003) was added. For Akt2/PKBβ,lysates were mixed in NETN with anti-Akt-2-agarose(Upstate-Biotechnology #16-174) and for Akt3/PKBγ, lysates were mixed inNETN with anti-Akt-3 agarose (Upstate Biotechnology #16-175). The IPswere incubated overnight at 4° C., washed and seperated by SDS-PAGE.

[0182] Western blots were used to analyze total Akt, pThr308 Akt,pSer473 Akt, and downstream targets of Akt using specific antibodies(Cell Signaling Technology): Anti-Total Akt (cat. no. 9272), Anti-PhophoAkt Serine 473 (cat. no. 9271), and Anti-Phospho Akt Threonine 308 (cat.no. 9275). After incubating with the appropriate primary antibodydiluted in PBS+0.5% non-fat dry milk (NFDM) at 4° C. overnight, blotswere washed, incubated with Horseradish peroxidase (HRP)-taggedsecondary antibody in PBS+0.5% NFDM for 1 hour at room temperature.Proteins were detected with ECL Reagents (Amersham/Pharmacia BiotechRPN2134).

Example 8

[0183] Heregulin Stimulated Akt Activation

[0184] MCF7 cells (a human breast cancer line that is PTEN^(+/+)) wereplated at 1×10⁶ cells per 100 mM plate. When the cells were 70-80%confluent, they were refed with 5 ml of serum free media and incubatedovernight. The following morning, compound was added and the cells wereincubated for 1-2 hours, heregulin was added (to induce the activationof Akt) for 30 minutes and the cells were analyzed as described above.

Example 9

[0185] Inhibition Of Tumor Growth

[0186] In vivo efficacy of an inhibitor of the growth of cancer cellsmay be confirmed by several protocols well known in the art.

[0187] Human tumor cell lines-which exhibit a deregulation of the PI3Kpathway (such as LnCaP, PC3, C33a, OVCAR-3, MDA-MB-468 or the like) areinjected subcutaneously into the left flank of 8-0.12 week old femalenude mice (Harlan) on day 0. The mice are randomly assigned to avehicle, compound or combination treatment group. Daily subcutaneousadministration begins on day 1 and continues for the duration of theexperiment. Alternatively, the inhibitor test compound may beadministered by a continuous infusion pump. Compound, compoundcombination or vehicle is delivered in a total volume of 0.1 ml. Tumorsare excised and weighed when all of the vehicle-treated animals,exhibited lesions of 0.5-1.0 cm in diameter, typically 4 to 5.5 weeksafter the cells were injected. The average weight of the tumors in eachtreatment group for each cell line is calculated.

1 16 1 10 DNA Artificial Sequence Completely synthetic DNA Sequence 1ctgcggccgc 10 2 14 DNA Artificial Sequence Completely synthetic DNASequence 2 gtacgcggcc gcag 14 3 39 DNA Artificial Sequence Completelysynthetic DNA Sequence 3 cgcgaattca gatctaccat gagcgacgtg gctattgtg 39 433 DNA Artificial Sequence Completely synthetic DNA Sequence 4cgctctagag gatcctcagg ccgtgctgct ggc 33 5 24 DNA Artificial SequenceCompletely synthetic DNA Sequence 5 gtacgatgct gaacgatatc ttcg 24 6 45DNA Artificial Sequence Completely synthetic DNA Sequence 6 gaatacatgccgatggaaag cgacggggct gaagagatgg aggtg 45 7 45 DNA Artificial SequenceCompletely synthetic DNA Sequence 7 cccctccatc tcttcagccc cgtcgctttccatcggcatg tattc 45 8 36 DNA Artificial Sequence Completely syntheticDNA Sequence 8 gaattcagat ctaccatgag cgatgttacc attgtg 36 9 30 DNAArtificial Sequence Completely synthetic DNA Sequence 9 tctagatcttattctcgtcc acttgcagag 30 10 48 DNA Artificial Sequence Completelysynthetic DNA Sequence 10 ggtaccatgg aatacatgcc gatggaaagc gatgttaccattgtgaag 48 11 33 DNA Artificial Sequence Completely synthetic DNASequence 11 aagcttagat ctaccatgaa tgaggtgtct gtc 33 12 30 DNA ArtificialSequence Completely synthetic DNA Sequence 12 gaattcggat cctcactcgcggatgctggc 30 13 49 DNA Artificial Sequence Completely synthetic DNASequence 13 ggtaccatgg aatacatgcc gatggaaaat gaggtgtctg tcatcaaag 49 146 PRT Artificial Sequence Completely synthetic Amino Acid Sequence 14Glu Tyr Met Pro Met Glu 1 5 15 13 PRT Artificial Sequence Completelysynthetic Amino Acid Sequence 15 Gly Gly Arg Ala Arg Thr Ser Ser Phe AlaGlu Pro Gly 1 5 10 16 15 PRT Artificial Sequence Completely syntheticAmino Acid Sequence 16 Lys Lys Gly Gly Arg Ala Arg Thr Ser Ser Phe AlaGlu Pro Gly 1 5 10 15

What is claimed is:
 1. A compound of the formula A:

wherein R¹ represents phenyl, furyl, thienyl or pyridinyl, any of whichgroups may be optionally substituted with one, two or threesubstituents, independently selected from: a) halogen; b) C₁₋₄ alkyl; c)C₁₋₄ alkoxy; d) cyano; e) di(C₁₋₄ alkyl)amino; f) hydroxy; R² representsamino-C₁₋₆ alkyl, C₁₋₄ alkylamino-(C₁₋₆)alkyl, di(C₁₋₄alkyl)amino-(C₁-6)alkyl, hydroxy-(C₁₋₆)alkyl or C₁₋₄ alkoxy-(C₁₋₆)alkyl,any of which groups may be optionally substituted; R³ representshydrogen or C₁₋₆ alkyl; and R⁴ independently represents hydrogen,C₁₋₆-alkyl, halogen, HO— or C₁₋₆ alkyl-O; r is 0, 1 or 2; or apharmaceutically acceptable salt or steteoisomer thereof.
 2. Thecompound according to claim 1 of the formula A-I:

wherein R² is as defined with reference to formula A above; R⁴ isselected from: C₃₋₇ cycloalkyl and phenyl, any of which groups may beoptionally substituted; r is 0, 1 or 2; s is 0, 1, 2 or 3; and R⁵independently represents halogen, C₁₋₄ alkyl or C₁₋₆ alkoxy; or apharmaceutically acceptable salt or stereoisomer thereof.
 3. Thecompound according to claim 1 which is:2,2,N,N-tetramethyl-N-(3-phenyl-[1,2,4]triazolo[3,4-α]phthalazin-6-yl)-propane-1,3-diamineN′-[3-(4-Methoxy-phenyl)-[1,2,4]triazolo[4,3-α]phthalazin-6-yl)-2,2,N,N-tetramethyl-propane-1,3-diamineor a pharmaceutically acceptable salt thereof.
 4. A pharmaceuticalcomposition comprising a pharmaceutical carrier, and dispersed therein,a therapeutically effective amount of a compound of claim
 1. 5. Apharmaceutical composition comprising a pharmaceutical carrier, anddispersed therein, a therapeutically effective amount of a compound ofclaim
 3. 6. A method for treating cancer which comprises administeringto a mammal in need thereof a therapeutically effective amount of acomposition of claim
 4. 7. A method for treating cancer which comprisesadministering to a mammal in need thereof a therapeutically effectiveamount of a composition of claim
 5. 8. A pharmaceutical composition madeby combining the compound of claim 1 and a pharmaceutically acceptablecarrier.
 9. A process for making a pharmaceutical composition comprisingcombining a compound of claim 1 and a pharmaceutically acceptablecarrier.